Human insulin analogs and preparations containing them

ABSTRACT

Novel human insulin analogs exhibiting high biological activity and in which the amino acid residue Phe B25  is substituted by His or Tyr and moreover substitutions may optionally be present in one or more of the positions A4, A8, A17, A21, B9, B10, B12, B13, B21, B26, B27, B28, and B30, as well as the amino acid residue in the B30 position may further be totally missing or may be blocked at the C-terminal in the form of ester or amide.

TECHNICAL FIELD

The present invention relates to novel human insulin analogs exhibitinga high specific biological activity as well as insulin preparationscontaining the human insulin analogs of the invention.

BACKGROUND ART

Ever since the discovery of insulin in 1922 many different types ofinsulin preparations have been used for the treatment of Diabetesmellitus. At the beginning exclusively insulin solutions exhibiting arapidly commencing and relatively rapidly ceasing insulin activity wereused, but later on insulin preparations exhibiting a wider profile ofactivity procured by lowering of the solubility of insulin by means ofadditions as e.g. zinc salt and/or protamines have been produced. Forreasons of availability the insulin used herefor has normally beenrecovered from Pancreas from domestic animals, most frequently oxes,pigs and sheep, however, recently preparations containing human insulinof biotechnological origin have also appeared on the market.

Throughout the years a large number of artificially prepared analogs ofhuman insulin has been described, usually with the purpose ofelucidating the influence of the structure on the activity, vide e.g.Marke et al., Hoppe-Seyler's Z. Physiol. Chem. 360, p. 1619-32 (1979).Investigations of the activity of substitutions of the(B22-B26)-sequence of the insulin on the receptor binding have been ofparticular interest, as said sequence is considered to be the main fieldof binding for the insulin receptor, and as naturally occurringmutations have been found with substitutions in said field. Vide e.g. S.Shoelson et al. PNAS 80, p. 7390-94 (1983) and M. Kobayashi et al.:Biomed. Res. 5 (3) p. 267-72 (1984). Very low activities for analogs inwhich Phe(B24) or Phe(B25) are replaced are thus found here, andtherefore it is concluded that the presence of these two amino acids isof decisive importance to the receptor binding.

Replacements in the insulin molecule can also be introduced with thepurpose of improving the profile of activity of the insulin in thetreatment of Diabetes. Thus, e.g. Danish Patent Application No. 5457/86discloses that one or more replacements of Glu in the insulin moleculeby a neutral amino acid residue causes a shifting of the zone ofprecipitation of the insulin in such a way that a slow release afterinjection is obtained.

Moreover, Danish Patent Application No. 4116/86 discloses insulinanalogs being particularly rapidly absorbed after injection. This effectis a result of the fact that by means of hydrophilic replacements inparticular in the B9and in the B28-positions in the insulin molecule asuppression of the aggregation ability of the insulin is obtained sothat it is essentially present as monomer. However, a number of theseinsulin analogs exhibits a reduced biological activity.

Peptides 1980, Proceedings of the Sixteenth European Peptide SymposiumHelsingor, Denmark, Aug. 31-Sep. 6, 1980, p. 372-377 describes thepreparation of [Tyr^(B25), Ala^(B30) ]-human insulin by enzymaticcoupling of the corresponding protected octapeptide to des-octapeptide(B23-B30) insulin followed by deprotection with trifluoroacetic acid. Nodata of biological activity is stated.

Biol. Chem. Hoppe-Seyler 1987, 368(6), p. 709-716 describes thepreparation of analogs of des-(B26-B30)- insulin-B25-amide in whichPhe^(B25) is replaced by Tyr^(B25) or His^(B25) thereby obtaining anincreased activity of 230 and 370%, respectively. The analogs aretruncated and at the B25 position.

DISCLOSURES OF THE INVENTION

It has now surprisingly been found that certain human insulin analogs inwhich Phe in position B25 is replaced by His or Tyr and optionallyfurther one or more of the amino acid residues in positions A4, A8, A17,A21, B9, B10, B12, B13, B21, B26, B27, B28, and B30 is replaced byanother amino acid residue, and in which the amino acid residue inposition B30 moreover may be missing totally or be blocked at theC-terminal in the form of ester or amide, exhibit a higher biologicalactivity than in such case where Phe in position B25 is unchanged.

Accordingly, the present invention relates to human insulin analogs inwhich the amino acid residue in position B25 is His or Tyr, the aminoacid residue in one or more of the positions A4, A8, A17, A21, B9, B10,B12, B13, B21, B26, B27, B28, and B30 is optionally replaced by anotheramino acid residue, and the amino acid residue in the B30-position isoptionally missing or blocked at the C-terminal in the form of ester oramide, provided that when B25 is Tyr then B30 is different from Ala.

Examples of particularly preferred substitutions in the above indicatedpositions are: A4:Gln, A8:His, A17:Gln, A21:Asp, B9:Asp, B10:Asp,B12:IIe, B13:Gln or Arg, B21:Gln or Ile, B26:Glu, B27:Arg, B28:Asp, andB30:Ala or Ser, provided that when B25 is Tyr then B30 is different fromAla. If several substitutions are desired at the same time in thepositions indicated above, it is preferred for reasons of use that theyare present within the group: A4, A8, A17, B13, B21, B27, and B30, orwithin the group: A8, A21, B9, B10, B12, B26, B28, and B30, providedthat when B25 is Tyr then B30 is different from Ala.

Thus, the human insulin analogs of the invention are advantageous in thetreatment of Diabetes, as the increased biological activity resultingfrom the replacements in the B25-position means a more rapid absorptionfrom the blood and moreover may neutralize the decrease of biologicalactivity which may otherwise be the result of other substitutions in thehuman insulin molecule.

A particular embodiment of the invention is represented by human insulinanalogs containing [His^(B25) ] or [Tyr^(B25) ] and at least onesubstitute amino acid residue selected from the group consisting of[Gln^(A4) ], [His^(A8) ], [Gln^(A17) ], [Asp^(A21) ], [Asp^(B9) ],[Asp^(B10) ], [Ile^(B12) ], [Gln^(B13) ], [Arg^(B13) ], [Gln^(B21) ],[Pro^(B21) ], [Glu^(B26) ], [Arg^(B27) ], [Asp ^(B28) ], [Ala^(B30) ],and [Ser^(B30) ], provided that when B25 is Tyr then B30 is differentfrom Ala.

Another particular embodiment of the invention is represented by humaninsulin analogs containing [His^(B25) ] or [Tyr^(B25) ] and at least twosubstitute amino acid residues selected from the group consisting of[Gln^(A4) ], [His^(A8) ], [Gln^(A17) ], [Gln^(B13) ], [Arg^(B13) ],[Gln^(B21) ], [Ile^(B21) ], [Arg^(B27) ], [Ala^(B30) ], and [Ser^(B30)], provided that when B25 is Tyr then B+is different from Ala.

Yet another particular embodiment of the invention is represented byhuman insulin analogs containing [His^(B25) ] or [Tyr^(B25) ] and atleast two substitute amino acid residues selected from the groupconsisting of [His^(A8) ], [Asp^(A21) ], [Asp^(B9) ], [Asp^(B10) ],[Ile^(B12) ], [Glu^(B26) ], [Asp^(B28) ], [Ala^(B30) ], and [Ser^(B30)], provided that when B25 is Tyr then B30 is different from Ala.

Preferred human insulin analogs of the invention are

[Tyr^(B25) ]-human insulin

[Tyr^(B25), Asp^(B28) ]-human insulin

[His^(B25) ]-human insulin

[His^(B25), Asp^(B28) ]-des[Thr^(B30) ]-human insulin

[Tyr^(B25) ]-human insulin-^(B30) -amide

[His^(B25) ]-human insulin-^(B30) -amide.

The described insulin derivatives can be produced biosynthetically inyeast expressing a DNA-sequence encoding a given insulin precursor.After the biosynthesis this precursor can be converted into the insulinderivative by an enzymatically catalyzed reaction. To achieve secretionto the growth medium, the DNA-sequence encoding the insulin precursorcan be fused to another DNA-sequence encoding a signal peptidefunctional in yeast. Secretion can be achieved by insertion in theexpression plasmid of the Saccharomyces cerevisiae Mfα1-leader sequence(Kurjan & Herskowitz, Cell 30, 933-943 (1982). A preferred constructionuses the DNA-sequence encoding the entire MFα1-leader sequence includingthe dibasic site LysArg but excluding the peptide sequence GluAlaGluAlawhich is the substrate for the yeast protease DPAP (dipeptidylaminopeptidase). In that way, an efficient secretion of insulinprecursors with the correct N-terminal is achieved.

DNA-sequences encoding modified insulin precursors were constructed withbasis in the expression cassette which is contained in the BamHIrestriction fragment from the expression plasmid pYGABA as shown in FIG.1, has a length of 1103 basepairs and contains essentially the following(listed in succession starting from the 5'-end): The GAPDH promoter(Travis et al., J. Biol. Chem., 260, 4384-4389 (1985) followed by thecoding region consisting of: The 83 N-terminal amino acids of theMFα1-leader sequence encoded by the wild-type yeast DNA sequence asdescribed by Kurjan & Herskowitz (reference given above) followed by thetwo codons AAA and AGA encoding Lys and Arg and again followed by thecoding region for the insulin precursor single chain des[Thr^(B30)]-human insulin (SCI), which is a synthetically constructed gene usingpreferred yeast codons. After two stop-codons, a SalI restriction siteis positioned, and the rest of the sequence constitutes theMFα1-sequence containing the terminator region. The sequence isconstructed using entirely standard techniques.

The method employed was "oligonucleotide site directed mutagenesis",which is described by Zoller & Smith, DNA, Vol. 3, No. 6, 479-488(1984). The method is briefly described in the following, and isdescribed thoroughly in Example 1. Isolated from the expression plasmidthe insulin precursor sequence is inserted into a single-stranded,circular M13 bacteriophage vector. To the single-stranded genom, achemically synthesized complementary DNA-strand is annealed. TheDNA-strand contains the desired sequence surrounded by sequencescompletely homologous to insulin sequences on the circular DNA. Invitro, the primer is then extended in the entire length of the circulargenom biochemically using Klenow polymerase. This strand will give riseto single-stranded phages, which when grown in E.coli give thepossibility to isolate double-stranded DNA with the desired sequence.From this double-stranded DNA, a restriction fragment can be isolatedand reinserted into the expression vector.

Human insulin analogs of the invention in which possible substitutionsare only present within the last amino acid residues nearest to theC-terminal of the B-chain may moreover in a manner known per se beprepared semisynthetically from e.g. porcine insulin as described in K.Inouye et al.: JACS 101 (3), p. 751-52 (1979), whereby the porcineinsulin is first split with trypsin to des-(B23-30)-human insulin,whereupon the latter, also enzymatically, is coupled with a syntheticpeptide having the desired amino acid sequence.

The invention also relates to insulin preparations which besides theusual adjuvants, excipients and/or carriers comprise at least one humaninsulin analog in which the amino acid residue in position B25 is His orTyr, the amino acid residue in one or more of the positions A4, A8, A17,A21, B9, B10, B12, B13, B21, B26, B27, B28, and B30 is optionallysubstituted, and the amino acid residue in the B30-position isoptionally missing or blocked at the C-terminal in the form of ester oramide. The insulin preparations of the invention may be preparedaccording to conventional methods for preparing insulin preparations.

MODES FOR CARRYING OUT THE INVENTION

The invention is further illustrated by the following Examples.

EXAMPLE I

Construction of an expression plasmid, which can be used to express[Tyr^(B25) ]-SCI.

The expression cassette, which is contained in the expression plasmidpYGABA (shown in FIG. 1) on a BamHI restriction fragment, was isolated:The expression plasmid was incubated with the restriction endonucleaseBamHI. The conditions were: 20 μg of plasmid, 50 units of BamHI, 100 mMNaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, and 1 mM DTT in a volume of100 μliters. The temperature was 37° C. and the reaction time 2 hours.The two DNA-fragments were separated on a 1% agarose gel, and thedesired fragment was isolated.

Ligation to the M13 vector M13mp18:

The isolated restriction fragment was ligated to the bacteriophagevector M13mp18 also cut with the restriction endonuclease BamHI in thefollowing reaction mixture: Fragment 0.2 μg, vector 0.02 μg, 50 mMTris-HCl, pH 7.4, 10 mM MgCl₂, 10 mM DTT and 1 mM ATP in a volume of 20μliters. 5 μliters of this mixture were transformed into the E. colistrain JM101. The presence of fragment in the vector and the orientationof the fragment was determined by restriction enzyme mapping ondouble-stranded M13-DNA isolated from the transformants.

Transformation of JM101:

The reaction mixture above was transformed in different dilutions intoCaCl₂ -treated E.coli JM101 cells using standard techniques and platedin 2×YT topagar on 2×YT agar plates. (2×YT=tryptone 16 g/liter, yeastextract 10 g/liter, NaCl 5 g/liter. 2×YT topagar=2×YT with 0.4% agaroseadded and autoclaved. 2×YT agar plates=2×YT with 2% agar added andautoclaved). The plates were incubated at 37° C. overnight.

Identification of positive clones:

The method used was plaque-lift hybridisation which is described in thefollowing: a nitrocellulose-filter was placed on a plate with a suitableplaque-density, so that the filter was wetted. The filter was thenbathed in the following solutions: 1.5M NaCl, 0.5 M NaOH for 30 sec.,1.5M NaCl, 0.5M Tris-HCl, pH 8.0 for 1 min., 2×SSC (0.3M NaCl, 0.03 Msodium citrate) till later use. The filter was dried on 3 MM filterpaper and baked for 2 hours at 80° C. in a vacuum oven.

The mutagenisation primer with the sequence 5'-TGGAGTGTAGTA-GAAACCTCT-3'was labelled radioactively in the 5' end in a 30 μliters volumecontaining 70 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 5 mM DTT, 10 pmololigonucleotide 20 pmol γ-³² P-ATP and 3.5 units of T4 polynucleotidekinase. The mixture was incubated at 37° C. for 30 min. and then for 5min. at 100° C.

The dried filter was pre-hybridised for 2 hours at 65° C. in 6×SSC, 0.2%bovine-serum albumin, 0.2% Ficoll, 0.2% polyvinylpyrrolidon, 0.2%sodium-dodecyl-sulphate (SDS) and 50 μg/ml salmon-sperm DNA. Then, thereaction mixture containing the labelled probe was added to 15 ml offresh pre-hybridisation mix, and the filter was bathed herein overnightat 31° C. with gentle shaking. After hybridisation, the filter waswashed 3 times for each 15 min. in 2×SSC+0.1% SDS and autoradiographed.After wash in the same solution, but now at 61° C., and anotherautoradiography, plaques containing DNA-sequences complementary to themutagenisation primer were identified.

Re-screening of positive clones:

Because the identified clone is a result of a heteroduplex, the plaquewas plated again. The hybridisation and identification were repeated.

Purification of double-stranded M13-phage DNA:

A re-screened clone was used for infection of the E. coli strain JM101.A culture containing approximately 10⁸ phages and 5 colonies of JM101was grown for 5 hours in a 5 ml 2×YT medium at 37° C. Then,double-stranded, circular DNA was purified from the pellet according toa method described by Birnboim & Doly, Nucleic Acids Res., 2, 1513(1979).

Isolation of single-stranded (ss) DNA (template):

From the transformant described above ss-DNA was isolated according to amethod described by Messing in Gene, 19, 269-276 (1982).

5'-phosphorylation of the mutagenisation primer:

The mutagenisation primer with the sequence 5'-TGGAGTGTAGTA-GAAACCTCT-3'was phosphorylated in the 5'-end in a 30 μliters reaction mixturecontaining 70 mM Tris-HCl, pH 7.0, 10 mM MgCl₂, 5 mM DTT, 1 mM ATP, 100pmol oligonucleotide and 3.6 units of T4 polynucleotide kinase. Thereaction was carried out for 30 min. at 37° C. Then, the enzyme wasinactivated by incubating the mixture for 10 min. at 65° C.

Annealing of template and mutagenisation primer:

Annealing of template and primer was carried out in a 10 μliters volumecontaining 0.5 pmol template, 4 pmol primer, 20 mM Tris-HCl, pH 7.5, 10mM MgCl₂, 50 mM NaCl and 1 mM DTT by heating for 10 min. at 65° C. andcooling afterwards to 0° C.

Extension/ligation reaction:

To the reaction mixture above, 10 μliters of the following mixture wereadded: 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.3 mM TTP, 1 mM ATP, 20mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 3 units of T4 DNA ligaseand 2.5 units of Klenow polymerase. Then, the reaction was carried outfor 16 hours at 16° C.

Isolation of a restriction fragment containing modified insulinprecursor:

The DNA-preparation (appr. 5 μg) isolated above was digested with 10units of the restriction endonuclease BamHI in 60 μliters of 100 mMNaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, and 1 mM DTT for 2 hours at37° C. The DNA-products were separated on an agarose-gel, and thefragment was purified from the gel.

Ligation to the yeast vector pAB24 (FIG. 2):

The isolated restriction fragment was ligated to the yeast vector pAB24digested with the restriction endonuclease BamHI in the followingreaction mixture: Fragment 0.2 μg, vector 0.02 μg, 50 mM Tris-HCl, pH7.4, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP in a total volume of 20 μliters. 5μliters of this reaction mix was used for transformation of the E. colistrain MC1061, in which the modified expression plasmid was identifiedand propagated. The plasmid was identical to pYGABA, except for thechanged codon.

Transformation of yeast:

Transformation of the expression plasmid into the yeast strainSaccharomyces cerevisiae JC482ΔpepΔLeu2ciro^(o) (α, his4, ura3, leu2,cir^(o)) was carried out as described by Ito et al., J. Bact., Vol. 153,No. 1, 163-168 (1983). The transformed cells were plated on SC-uramedium (0.7% Yeast Nitrogen Base, 2.0% glucose, 0.5% casamino acids,2.0% agar) for selection for plasmid-containing cells.

EXAMPLE II

Construction of an expression plasmid, which can be used for productionof [His^(B25), Asp^(B28) ]-SCI.

The procedure used was essentially the same as described in Example I,except that the mutagenisation primer had the sequence5'ACAATACCCTTGTCAGTGTAGTGGAAACCTCTTT3', that the hybridisationtemperature was 42° C., and that the washing temperature after thehybridisation was 63° C. The modified plasmid had a sequence identicalto pYGABA, except for the modified codons.

EXAMPLE III

Expression of precursor and isolation from the culture medium.

Yeast, transformed as described in Example I or II, was propagated onPetri-plates containing minimal-medium without uracil for 48 hours at30° C. 100 ml shake bottles containing minimal-medium without uracil+5g/liter casamino acids+10 g/liter succinic acid+30 g/liter glucose at pH5.0 was inoculated with a single colony from the Petri-plate. Thebottles were then shaken at 30° C. in incubator for 72 hours.

After centrifugation 1 liter of pooled supernatant was sterilized byfiltration and adjusted to pH 4-4.5 and a conductivity<10 mS by additionof 5M HCl and water. With a flow of 120 ml/hour the supernatant was thenapplied to a 1.6×6 cm column of S-Sepharose® FF previously equilibratedwith 50 mM acetic acid, 50% (by volume) ethanol adjusted to pH 4.0 withNaOH. The column was washed with 60 ml buffer and the precursor was theneluted by a linear gradient of NaCl from 0 to 0.35 M in 360 ml bufferwith a flow of 10 ml/hour. The eluate was divided in fractions of 4 mland detected for UV-absorbance. Fractions containing precursor wereidentified by RP-HPLC analysis and were pooled. After desalting on acolumn of Sephadex® G25 in 1 M acetic acid the precursor was isolated bylyophilization.

EXAMPLE IV

Preparation of [Tyr^(B25) ]-des[Thr^(B30) ]-human insulin fromprecursor.

450 mg of [Tyr^(B25) ]-SCI, prepared by the methods described inExamples I and III, were dissolved in 45 ml of 50 mMtris(hydroxymethyl)aminomethane, 20% (by volume) ethanol adjusted to pH7.7 with HCl and 45 ml (settled volume) of Sepharose® containing 36 mgof immobilized trypsin in the same buffer were added. The suspension wasleft for 3 hours at 20° C. with gentle agitation and was then filtered.The gel was washed with 40 ml of buffer, and the pooled filtrates wereapplied to a 2.6×7.5 cm column of Q-Sepharose® FF previouslyequilibrated with 50 mM tris(hydroxymethyl)aminomethane, 50% (by volume)ethanol, adjusted to pH 8.0 with HCl. The column was then eluted with alinear gradient of NaCl from 0 to 0.15M in the same buffer over 6 hourswith a flow of 225 ml/hour. The eluate was detected for UV-absorbanceand fractions containing the main protein peak were pooled.

After dilution with the same volume of water the pool was applied to a2.6×25 cm column of Lichroprep® RP-18 (25-40 μm) previously equilibratedwith 10 mM H₃ PO₄, 0.1M NaCl, 30% (by volume) ethanol. With a flow of 16ml/hour the column was then eluted with a linear gradient of ethanolfrom 30% to 40% (by volume) over 20 hours. The eluate was detected forUV-absorbance and fractions containing the main protein peak werepooled. After desalting on a column of Sephadex® G25 in 1M acetic acid105 mg of [Tyr^(B25) ]-des[Thr^(B30) ]-human insulin were isolated bylyophilization.

The protein was redissolved in a mixture containing 200 mg of threoninemethyl ester, 1.0 ml of ethanol and 0.4 ml of water. The pH value wasadjusted to 6.3 with acetic acid, and 2 ml (settled volume) ofSepharose® containing 1.6 mg of immobilized trypsin were added. Afterstanding for 2 hours at 20° C. with gentle agitation, the gel wasremoved by filtration, and the protein was precipitated by addition of10 volumes of 2-propanol. The air-dried precipitate was redissolved in20 mM tris(hydroxymethyl)aminomethane/HCl, 60% (by volume) ethanol, pH8.25, and applied to a 1.6×20 cm Q-Sepharose® FF column, previouslyequilibrated with the said buffer, and eluted with a linearNaCl-gradient in the same buffer increasing from 0 to 0.1M over 15 hoursat a flow rate of 50 ml/hour. The ethanol was removed in vacuo from thepooled fractions containing [Tyr^(B25) ] human insulin-(B30-methylester), and the protein was precipitated by adjusting the pH value to6.1. After centrifugation and lyophilization the methyl ester washydrolyzed for 10 min. in cold 0.1M NaOH at a protein concentration of10 mg/ml. The reaction was stopped by adjusting the pH value to 8.5, and2 volumes of 20 mM tris(hydroxymethyl)aminomethane/HCl, pH 8.5, wereadded. The solution was then applied to a 1.6×20 cm Q-Sepharose® FFcolumn and eluted as described above. The protein was precipitated at apH value of 5.5 after removal of the ethanol in vacuo. 40 mg of[Tyr^(B25) ]-human insulin were obtained after lyophilization.

The identity of the product was confirmed by amino acid analysis and bysequential Edman degradation of the separated vinylpyridylated A- andB-chains.

EXAMPLE V

Preparation of [His^(B25), Asp^(B28) ]-des[Thr^(B30) ]-human insulinfrom precursor.

100 mg of [His^(B25), Asp^(B28) ]-SCI, prepared by the methods describedin Examples II and III, were dissolved in 10 ml of 50 mMtris(hydroxymethyl)aminomethane, 20% (by volume) ethanol, adjusted to pH7.7 with HCl and 10 ml (settled volume) of Sepharose®, containing 8 mgof immobilized trypsin, in the same buffer were added. The suspensionwas left for 3 hours at 20° C. with gentle agitation and was thenfiltered. The gel was washed with 10 ml of buffer, and the pooledfiltrates were applied to a 1.6×7.5 cm column of Q-Sepharose® FFpreviously equilibrated with 50 mM tris(hydroxymethyl)aminomethane, 50%(by volume) ethanol, adjusted to pH 8.0 with HCl. The column was theneluted with a linear gradient of NaCl from 0 to 0.15M in the same bufferover 6 hours with a flow of 90 ml/hour. The eluate was detected forUV-absorbance, and fractions containing the main protein peak werepooled.

After dilution with the same volume of water the pool was applied to a1.6×25 cm column of Lichroprep® RP-18 (25-40 um) previously equilibratedwith 10 mM H₃ PO₄, 0.1M NaCl, 30% (by volume) ethanol. With a flow of6.5 ml/hour the column was then eluted with a linear gradient of ethanolfrom 30% to 40% (by volume) over 20 hours. The eluate was detected forUV-absorbance, and fractions containing the main protein peak werepooled. After desalting on a column of Sephadex® G25 in 1M acetic acid39 mg of [His^(B25), Asp^(B28) ]-des[Thr^(B30) ]-human insulin wereisolated by lyophilization.

The identity of the product was confirmed by amino acid analysis and bysequential Edman degradation of the separated vinylpyridylated A- andB-chains.

EXAMPLE VI

Preparation of des-(B23-B30)-human insulin.

1 g of Na-crystallized porcine insulin (protein weight) was dissolved in40 ml of water, and a solution of 50 mg of porcine trypsin in 10 ml offreshly prepared 0.25M ammonium hydrogencarbonate solution in which pHhas been adjusted to 9.0 with ammonia water was added. Then, thesolution was left in a refrigerator at 4° C. After 48 hours a HPLCanalysis showed a degree of reaction of 65%. Thereafter the solution wasgel-filtered at 4° C. on a 5×90 cm column of Sephadex® G50 Superfine in0.05M ammonium hydrogencarbonate at a flow of 90 ml/hour. Fractionscontaining the main protein peak were pooled and lyophilized.

Yield: 520 mg of des-(B23-30)-human insulin of a purity of 97.5%,measured by HPLC analysis.

EXAMPLE VII

Preparation of peptides.

Peptides were prepared by means of a peptide synthesis machine fromApplied Biosystems, whereby the construction was made on a PAM resin bymeans of protected symmetrical amino acid anhydrides. The capacity wasabout 0.1 mmole of peptide. Finally, the peptide was split from theresin by reaction with anhydrous hydrogen fluoride at 0° C., whereby theremaining protection groups were simultaneously removed.

EXAMPLE VIII

Preparation of [His^(B25) ]-human insulin.

200 mg of des-(B23-30)-human insulin and 400 mg ofGly-Phe-His-Tyr-Thr-Pro-Lys-Thr were dissolved in a mixture of 2.40 mlof dimethyl formamide and 1.20 ml of water, the pH of the mixture beingadjusted to 6.5 with triethyl amine.

Then, a solution of 10 mg of porcine trypsin in 0.20 ml of water wasadded, and the reaction mixture was left at 20° C. After 4 hours a HPLCanalysis showed a reaction of 60%, and the reaction was stopped byprecipitation with 25 ml of 2-propanol. The precipitate which wasisolated by centrifugation was redissolved in 10 ml of 1M acetic acidand applied at 20° C. to a 2.6×20 cm column of Lichroprep® RP-18 (25-40μm), equilibrated in a buffer consisting of 0.5 mM hydrogenchloride,0.1M sodium chloride in 30% (by volume) ethanol. Then the column waseluted with the same buffer at a flow of 20 ml/hour, the content ofethanol however being linearly increased to 50% over 24 hours. Thefractions containing the main protein peak were pooled, whereupon theprotein was precipitated by dilution with the same volume of water andadjustment of pH to 5.5 with sodium hydroxide solution. After standingat 4° C. for 1 hour the suspension was centrifuged, and the precipitatewas freeze-dried. Hereby 90 mg of [His^(B25) ]-human insulin wereobtained, identified by total amino acid analysis and by sequentialEdman degradation of the separated vinylpyridylated A- and B-chains.

EXAMPLE IX

Preparation of [Tyr^(B25), Asp^(B28) ]-human insulin.

150 mg of des-(B23-30)-human insulin and 350 mg ofGly-Phe-Tyr-Tyr-Thr-Asp-Lys-Thr were dissolved in a mixture of 2.0 ml ofdimethyl formamide and 1.0 ml of water, the pH being adjusted to 6.5with triethyl amine. Now a solution of 8 mg of porcine trypsin in 0.20ml of 1 mM calcium acetate solution was added, and the reaction mixturewas left at 15° C. After 3 hours a HPLC analysis showed a reaction of50%, and the reaction was stopped by adding 25 ml of 2-propanol.

After centrifugation the precipitate was redissolved in 10 ml of 1Macetic acid and applied at 20° C. to a 2.6×20 cm column of Lichroprep®RP-18 (25-40 μm), equilibrated in a buffer consisting of 0.5 mMhydrogenchloride, 0.1M sodium chloride in 30% (by volume) ethanol. Then,the column was eluted with the same buffer at a flow of 20 ml/hour, thecontent of ethanol however being linearly increased to 50% over 24hours. The fractions containing the main protein peak were pooled,whereupon the protein was precipitated by dilution with the same volumeof water and adjustment of pH to 5.0 with sodium hydroxide solution.After standing overnight at 4° C. the precipitate was isolated bycentrifugation and freeze-dried.

Yield: 60 mg of [Tyr^(B25), Asp^(B28) ]-human insulin, identified bytotal amino acid analysis and by sequential Edman degradation of theseparated vinylpyridylated A- and B-chains.

EXAMPLE X

Evaluation of biological activity.

The biological activity in vitro was determined by measuring the bindingaffinity to the insulin receptors of isolated rat adipocytes andhepatocytes essentially as described in J. Gliemann, S. Gammeltoft:Diabetologia 10, 105-113 (1974).

The insulin analogs were compared to semisynthetic human insulin, thepotency of which was set to 100%, and the results are shown in the Tablebelow:

    ______________________________________                                                         Adipocytes                                                                             Hepatocytes                                         ______________________________________                                        [Tyr.sup.B25 ]-human insulin                                                                     356 %      222%                                            [Tyr.sup.B25, Asp.sup.B28 ]-human insulin                                                        201%       138%                                            [His.sup.B25 ]-human insulin                                                                     150%       142%                                            [His .sup.B25, Asp.sup.B28 ]-des[Thr.sup.B30 ]-human                                             130%       135%                                            insulin                                                                       ______________________________________                                    

We claim:
 1. Human insulin analogs exhibiting high biological activity, characterized in that the amino acid residue in position B25 is His or Tyr, that the amino acid residue in one or more of the positions A4, A8, A17, A21, B9, B10, B12, B13, B21, B26, B27, B28, and B30 is optionally replaced by another amino acid residue, and that the amino acid residue in the B30-position is optionally missing or blocked at the C-terminal in the form of ester or amide, provided that when B25 is Tyr then B30 is different from Ala.
 2. Human insulin analogs according to claim 1, characterized by containing [His^(B25) ] or [Tyr^(B25) ] and at least one substitute amino acid residue selected from the group consisting of [Gln^(A4) ], [His^(A8) ], [Gln^(A17) ], [Asp^(A21) ], [Asp^(B9) ], [Asp^(B10) ], [Ile^(B12) ], [Gln^(B13) ], [Arg^(B13) ], [Gln^(B21) ], [Pro^(B21) ], [Glu^(B26) ], [Arg^(B27) ], [Asp^(B28) ], [Ala^(B30) ], and [Ser^(B30) ], provided that when B25 is Tyr then B30 is different from Ala.
 3. Human insulin analogs according to claim 1, characterized by containing [^(B25) ] or [Tyr^(B25) ] and at least two substitute amino acid residues selected from the group consisting of [Gln^(A4) ], [His^(A8) ], [Gln^(A17) ], [Gln^(B13) ], [Arg^(B13) ], [Gln^(B21) ], [Ile^(B21) ], [Arg^(B27) ], [Ala^(B30) ], and [Ser^(B30) ], provided that when B25 is Tyr then B30 is different from Ala.
 4. Human insulin analogs according to claim 1, characterized by containing [His^(B25) ] or [Tyr^(B25) ] and at least two substitute amino acid residues selected from the group consisting of [His^(A8) ], [Asp^(A21) ], [Asp^(B9) ], [Asp^(B10) ], [Ile^(B12) ], [Glu^(B26) ], [Asp^(B28) ], [Ala^(B30) ], and [Ser^(B30) ], provided that when B25 is Tyr then B30 is different from Ala.
 5. Human insulin analog according to claim 1, characterized by being [Tyr^(B25) ]-human insulin.
 6. Human insulin analog according to claim 1, characterized by being [Tyr^(B25), Asp^(B28) ]-human insulin.
 7. Human insulin analog according to claim 1, characterized by being [His^(B25) ]-human insulin.
 8. Human insulin analog according to claim 1, characterized by being [His^(B25), Asp^(B28) ]-des-[Thr^(B30) ]-human insulin.
 9. Human insulin analog according to claim 1, characterized by being [Tyr^(B25) ]-human insulin-B30-amide.
 10. Human insulin analog according to claim 1, characterized by being [His^(B25) ]-human insulin-B30-amide.
 11. Insulin preparation, characterized by containing at least one human insulin analog in which the amino acid residue in position B25 is His or Tyr, the amino acid residue in one or more of the positions A4, A8, A17, A21, B9, B10, B12, B13, B21, B26, B27, B28, and B30 is optionally replaced by another amino acid residue, and the amino acid residue in the B30-position is optionally missing or blocked at the C-terminal in the form of ester or amide, provided that when B25 is Tyr then B30 is different from Ala.
 12. Insulin preparation according to claim 11, characterized by containing at least one human insulin analog comprising [His^(B25) ] or [Tyr^(B25) ] and at least one substitute amino acid residue selected from the group consisting of [Gln^(A4) ], [His^(A8) ], [Gln^(A17) ], [Asp^(A21) ], [Asp^(B9) ], [Asp^(B10) ], [Ile^(B12) ], [Gln^(B13) ], [Arg^(B13) ], [Gln^(B21) ], [Ile^(B21) ], [Glu^(B26) ], [Arg^(B27) ], Asp^(B28) ], [Ala^(B30) ], and [Ser^(B30) ].
 13. Insulin preparation according to claim 11, characterized by containing at least one human insulin analog comprising [His^(B25) ] or [Tyr^(B25) ] and at least two substitute amino acid residues selected from the group consisting of [Gln^(A4) ], [His^(A8) ], [Gln^(A17) ], [Gln^(B13) ], [Arg^(B13) ], [Gln^(B21) ], [Ile^(B21) ], [Arg^(B27) ], [Ala^(B30) ] and [Ser^(B30) ].
 14. Insulin preparation according to claim 11, characterized by containing at least one human insulin analog comprising [His^(B25) ] or [Tyr^(B25) ] and at least two substitute amino acid residues selected from the group consisting of [His^(A8) ], [Asp^(A21) ], [Asp^(B9) ], [Asp^(B10) ], [Ile^(B12) ], [Glu^(B26) ], [Asp^(B28) ], [Ala^(B30) ], and [Ser^(B30) ].
 15. Insulin preparation according to claim 11, characterized by containing [Tyr^(B25) ]-human insulin.
 16. Insulin preparation according to claim 11, characterized by containing [Tyr^(B25), Asp^(B28) ]-human insulin.
 17. Insulin preparation according to claim 11, characterized by containing [His^(B25) ]-human insulin.
 18. Insulin preparation according to claim 11, characterized by containing [His^(B25), Asp^(B28) ]-des-[Thr^(B30) ]-human insulin.
 19. Insulin preparation according to claim 11, characterized by containing [Tyr^(B25) ]-human insulin-B30-amide.
 20. Insulin preparation according to claim 11, characterized by containing [His^(B25) ]-human insulin-B30-amide. 